Throughout this application, various publications are referenced and citations provided for them. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
It is now a well established fact that all living organisms including infectious agents, e.g., viruses, contain DNA, or sometimes RNA, molecules which carry genetic information in the form of a nucleotide sequence code. While certain segments of this code are shared by many organisms, there are other segments which contain nucleotide sequences that are unique for a particular organism. These sequences are said to be species-specific and provide a convenient tag or footprint that can be utilized for identification of that organism. The technique of nucleic acid hybridization (Gillespie and Spiegelman, 1965) has great potential for the rapid detection and typing of infectious agents. However, current hybridization assays have not yet attained the sensitivity and speed required for practical diagnostic use. It has recently been proposed that the sensitivity and speed of bioassays could be improved by linking a replicatable RNA to a hybridization probe (Chu, et al. 1986). After hybridization, the replicatable RNA would be amplified by incubation with the RNA-directed RNA polymerase, Qxcex2 replicase (Haruna and Spiegleman, 1965a). The enormous number of RNA copies that would be synthesized would serve as a signal that hybridization had occurred. The synthesis of novel nucleic acid hybridization probes that combine in a single RNA molecule the dual functions of probe and amplifiable reporter is described in this invention.
A distinguishing feature of RNA synthesis by Qxcex2 replicase is that a small number of template strands can initiate the synthesis of a large number of product strands (Haruna and Spiegleman, 1965b). Million-fold increases in the amount of RNA routinely occur in vitro (Kramer, et al. 1974) as a result of an autocatalytic reaction mechanism (Weissman, et al. 1986; Spiegelman, et al. 1968): single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products; after the completion of product strand elongation, both the product and the template are released from the replication complex (Dobkin, et al. 1979); and both strands are free to serve as templates in the next round of synthesis. Consequently, as long as there is an excess of replicase, the number of RNA strands increases exponentially. After the number of RNA strands equals the number of active replicase molecules, RNA synthesis continues linearly.
Qxcex2 replicase was first isolated from bacteriophage Qxcex2-infected Escherichia coli by Haruna and Spiegelman (1965a). It is composed of four polypeptides, only one of which is specified by the viral RNA. The other three polypeptides are E. coli proteins, and have been identified as the protein synthesis elongation factors Tu and Ts and the ribosomal protein S1. When provided with the single-stranded RNA from Qxcex2, the replicase mediates the exponential synthesis of infectious viral RNA (Spiegelman et al., 1965). The enzyme is highly template selective. No other viral RNA, nor any E. coli RNA, will serve as a template (Haruna and Spiegelman, 1965c). When RNA from a temperature-sensitive mutant of Qxcex2 was used as a template with wild-type replicase, mutant RNA was synthesized, demonstrating that the template is the instructive agent (Pace and Spiegelman, 1966). The replicative process (Spiegelman et al., 1969; Weissmann et al., 1968) proceeds in the following manner: The replicase uses the viral (+) strand as a template to direct the synthesis of a complementary (xe2x88x92) strand. Both of these strands serve as templates for the synthesis of additional (+) and (xe2x88x92) strands; and exponential increase is observed in the number of RNA strands present. Eventually, there are enough strands to saturate the available enzyme molecules, after which the number of strands increases linearly with time. Because of the complementary nature of this process, it is often referred to as xe2x80x9cself-replicationxe2x80x9d. There are a number of advantages to using the amplification of RNA by Qxcex2 replicase as the basis of a signal-generating system: Qxcex2 replicase is highly specific for its own template RNAs (Haruna and Spiegelman, 1965c); as little as one molecule of template RNA can, in principle, initiate replication (Levisohn and Spiegelman, 1968); and the amount of RNA synthesized (typically, 200 ng in 50 xcexcl in 15 minutes) is so large that it can be measured with the aid of simple colorimetic techniques. There are a number of naturally occurring Qxcex2 replicase templates that are much smaller than Qxcex2 RNA. These RNAs have been isolated from in vitro Qxcex2 replicase reactions that were incubated in the absence of exogenous template RNA. They include: MDV-1 RNA (Kacian et al., 1972), microvariant RNA (Mills et al., 1975), the nanovariant RNAs (Schaffner et al., 1977), RQ120 RNA (Munishkin at al., 1989), and cordycepin-tolerant RNA (Priano et al., 1989). Although the origin and biological role of these RNAs is not known, they have been extensively characterized and are all excellent templates for Qxcex2 replicase.
Isolated MDV-1 RNA serves as an excellent exogenous template. It is bound by Qxcex2 replicase and replicated in a manner similar to Qxcex2 RNA (Kacian et al., 1972). MDV-1 RNA is much smaller (221 nucleotides) than Qxcex2 RNA (4,220 nucleotides), which led to the determination of its complete nucleotide sequence (Mills et al., 1973; Kramer and Mills, 1978).
Two striking aspects of the MDV-1 sequence are its unusually high proportion of guanosine and cytidine residues and the occurrence of many intrastrand complements capable of forming hairpin structures. MDV-1 has been directly visualized (Klotz et al., 1980), utilizing hollow-cone, dark-field electron microscopy. Observations made with native, partially denatured, and fully denatured molecules indicate that native single-stranded MDV-1 RNA is a highly condensed molecule, possessing substantial tertiary structure. Specific secondary structures were identified by reacting MDV-1 RNA with chemical agents that modify single-stranded regions (Mills et al., 1980). The location of the altered nucleotides was determined by sequencing the modified RNA. The tertiary structure of MDV-1 RNA was probed by subjecting it to mild cleavage with ribonuclease T1 (Kramer et al., 1989), which only cleaves single-stranded regions. Because of the extensive secondary and tertiary structure present in MDV-1 RNA, combined with the macromolecular dimensions of ribonuclease T1, the initial sites of attack were limited to those on the exterior of the molecule. The few guanosines in each strand that were hypersusceptible to ribonuclease T1 were located in hairpin loops.
A more detailed understanding of the mechanism of MDV-1 RNA synthesis was facilitated by the development of an electrophoretic technique for separating the complementary strands (Mills et al., 1978). An excess of pure MDV-1 (xe2x88x92) RNA was used as template in the presence of a small amount of Qxcex2 replicase, in a series of experiments designed to elucidate the synthetic cycle. Mutant MDV-1 (xe2x88x92) RNA (Kramer et al., 1974) was added to these reactions after the initiation of chain elongation to see whether replicase molecules retain the same template through many rounds of synthesis. It was shown that a single replicase molecule bound to a single template strand is sufficient to carry out a complete synthetic cycle. It was shown that after the completion of each round of chain elongation, the product strand is released from the replication complex, and the template and the replicase then dissociate (Dobkin et al., 1979).
Specific regions of MDV-1 RNA are required by Qxcex2 replicase to carry out different replication functions. A highly structured region in the middle of each complementary strand must be present for the replicase to bind to MDV-1 RNA. Utilizing a series of different-length fragments of MDV-1 RNA that were missing sequences at either their 5xe2x80x2 end or their 3xe2x80x2 end, the binding region was localized by assaying their ability to be bound by the replicase (Nishihara et al., 1983). Only fragments containing the central recognition region formed a complex. This region contains hairpins that were found to be hypersusceptible to ribonuclease T1 cleavage (Kramer et al., 1989), and is therefore situated on the exterior of the molecule,
A particular sequence at 3xe2x80x2 end of the template strand is required for the initiation of product strand synthesis. MDV-1 RNA fragments lacking nucleotides at their 5xe2x80x2 end were able to serve as templates for the synthesis of complementary strands, but fragments lacking nucleotides at their 3xe2x80x2 end were unable to serve as templates, even though they were able to form complexes with the replicase (Nishihara et al., 1983). The sequence required for initiation to occur includes at least some of the 3xe2x80x2 terminal cytidines, since the conversion of these cytidines to uridines by sodium bisulfite treatment was accompanied by a concomitant loss in the ability of the RNA to initiate synthesis (Mills et al., 1980). In a control experiment, MDV-1 (+) RNA was completely modified by bisulfite treatment, except for the 3xe2x80x2 -terminal cytidines, which were protected by a complementary oligonucleotide mask. The resulting RNA was able to initiate synthesis. The 5xe2x80x2 end of the template need not be present for the synthesis of multiple copies of complementary RNA (Bausch et al., 1983).
However, it must be present if exponential synthesis is to occur, because the 5xe2x80x2 terminal sequence serves as the template for the required 3xe2x80x2 initiation sequence in the product strand.
An exhaustive comparison of the nucleotide sequence of MDV-1 RNA and Qxcex2 RNA (Nishihara et al., 1983) found only two significant homologies. These regions encompass the internal replicase binding site required for template recognition and the cytidine-rich 3xe2x80x2-terminal sequence required for product strand initiation.
Template secondary structures strongly influence the rate of chain elongation. Electrophoretic examination of the distribution of different-length elongation intermediates during the synthesis of MDV-1 RNA indicated that the rate of synthesis is highly variable (Mills et al., 1978). The data suggest that at a relatively small number of specific sites the progress of the replicase is temporarily interrupted, and then resumes spontaneously with a finite probability. The time spent at each of these pause sites is so long that the mean time it takes to replicate an RNA is well approximated by the sum of the mean times spent at each site. Nucleotide sequence analysis of the most prominent elongation intermediates indicated that each could form a strong 3xe2x80x2-terminal hairpin structure. This suggests that the marked variability in the rate of chain elongation is due to the formation of terminal hairpins in the product strand, or the reformation of hairpins in the template strand. Secondary structures that form during chain elongation are free to dissociate and form stronger secondary structures as nucleotides are added to the growing product strand (Kramer and Mills, 1981). Since the formation of secondary structures apparently slows down the rate of polymerization, their presence in the RNA, per se, is disadvantageous, and they should not have evolved. These structures must therefore confer a selective advantage that outweighs their negative effect on the rate of synthesis. Recent experiments that compared the replication of cordycepin-tolerant RNA to the replication of MDV-1 RNA and microvariant RNA have shown that the formation of these secondary structures during chain elongation is required to prevent the collapse of the product strand upon the template strand (Priano et al., 1987).
Many investigators have wished to exploit the autocatalytic nature of Qxcex2 replicase reactions to synthesize large amounts of any RNA in vitro. However, Qxcex2 replicase cannot copy most RNAS. It is highly selective for its own naturally occurring templates. Two specific interactions between the replicase and the template RNA account for this specificity. The replicase binds to the template at the internal recognition sequence, and the cytidine-rich sequence at the 3xe2x80x2 end of the template is required for the initiation of product strand synthesis. Each of these sequences must be present in an RNA for exponential synthesis to occur. The exponential synthesis of heterologous RNAs was achieved by constructing recombinant RNAs by the insertion of heterologous sequences at an appropriate site within MDV-1 RNA (Miele et al., 1983). The insertion site chosen (between nucleotides 63 and 64 of MDV-1 (+) RNA) was located away from the regions that are required for template recognition and product strand initiation. The site was in hairpin loop, where it was least likely to disturb structure; and it was in a loop where viable mutations were known to occur, indicating that the sequence in that region was not essential for replication. Furthermore, this site was located on the exterior of the molecule in a hairpin loop that was hypersusceptible to cleavage by ribonuclease T1. The first recombinant RNA constructed wag prepared by cleaving MDV-1 (+) RNA at the selected site with ribonuclease T1 and then inserting decaadenylic acid there by direct ligation with the aid of bacteriophage T4 RNA ligase. The resulting 231-nucleotide recombinant was an excellent template for Qxcex2 replicase, demonstrating that the inserted sequence did not interfere with replication. The products consisted of full-length copies of the recombinant RNA, and both complementary strands were synthesized. An analysis of the kinetics of recombinant RNA synthesis demonstrated that the amount of recombinant RNA increased exponentially at a rate that was indistinguishable from that of MDV-1 RNA.
A plasmid that contains a strong bacteriophage T7 promoter (Lizardi et al., Mills et al., 1990) directed towards the MDV-1 cDNA sequence is a good template for MDV-1 RNA synthesis (Axelrod and Kramer, 1985). Bacteriophage RNA polymerases are highly specific for their own promoters (Chamberlin and Ring, 1973). Incubation with a bacteriophage RNA polymerase results in the exclusive transcription of the nucleotide sequence immediately downstream from its homologous promoter (McAllister et al., 1981; Melton et al., 1984). Moreover, the transcripts are virtually homogenous; and as many as 300 transcripts can be synthesized from each promoter in a 30 minute incubation.
The two developments discussed supra led to the present invention, namely, the discovery that heterologous RNA segments can be inserted within the sequence of a small, naturally occurring template for Qxcex2 replicase, MDV-1 RNA (Kacian, et al. 1972), without affecting its replicability Miele, 1983 (Miele, et al. 1983); and the construction of a plasmid that serves as a template for the synthesis of MDV-1 (+) RNA when the plasmid is incubated in vitro with bacteriophage T7 RNA polymerase (Mills, et al. 1990).
This plasmid has been modified in the present invention by inserting a polylinker within the MDV-1 cDNA sequence, and then inserting synthetic hybridization probe sequences within the polylinker. The resulting plasmids served as templates for the synthesis of xe2x80x9crecombinant RNAs,xe2x80x9d which consist of a probe sequence embedded within the sequence of MDV-1 (+) RNA. The site for inserting the polylinker and probe into MDV-1 RNA is on the exterior of the molecule to reduce any possible interference with replication and hybridization of the probe sequence to its target. The probe sequences employed in this invention and which will be further described in the Experimental Details section to follow, are known to hybridize specifically to the repetitive DNA of Plasmodium falciparum (Franzen, et al. 1984; Aslund, et al. 1985; Zolg, et al. 1987), one of the protozoans that cause malaria, and to a conserved region of the HIV-1 pol gene. These recombinant RNA molecules are bifunctional, in that they are able to hybridize specifically to complementary DNA targets and they are also able to serve as templates for exponential amplification by Qxcex2 replicase.
Infectious agents may be quite rare in asymptomatic individuals who are infectious to others. For example, an asymptomatic individual may have as few as one in 100,000 peripheral blood mononuclear cells infected with the pathogenic retrovirus, human immunodeficiency virus type 1 (HIV-1), yet donated blood from that person will readily infect others (Harper, et al. 1986). It is thus imperative that very sensitive clinical assays be developed for detecting HIV-1, to screen donated blood and to identify asymptomatic carriers. Suitable assays would make use of a macromolecular probe having extremely high affinity for a particular component of the infections agent and very low affinity for all the other components of the sample. The highest specificities and most stable interactions known occur when a single-stranded oligonucleotide probe hybridizes to a complementary oligonucleotide target (Gillespie and Spiegelman, 1965). For example, oligonucleotide probes can seek out and bind to the integrated HIV-1 DNA, or the retroviral messenger RNA, present in a single infected cell.
However, the use of oligonucleotide probes is not sufficient to ensure detection. An infected cell contains only about 6000 molecules of retroviral messenger RNA (Pelligrino, et al. 1987), so the problem becomes how to detect the probes once they are bound to such a small number of targets. The classic detection strategy is to attach reporter groups to the probes, such as fluorescent organic molecules or radioactive phosphate groups. More recently, biotin groups have been incorporated into probes (Langer, et al. 1981). After the probes have bound to their targets, enzymes such as peroxidase or phosphatase are linked to the biotin, then incubated with a colorless substrate, leading to the accumulation of a large number of colored product molecules for each enzyme-probe adduct (Leary, et al. 1983). However, the practical limit of detection of these schemes is about 106 target molecules. Clearly, they cannot be used to detect a single cell in a sample that contains only 6000 retroviral messenger RNAS.
A particularly attractive strategy for detecting rare targets is to link each probe to a replicatable reporter, which can be exponentially amplified after hybridization to reveal the presence of the probe (Chu, et al. 1986). The present invention concerns a novel version of this approach, in which a probe sequence is embedded within the sequence of a replicatable RNA (Lizardi, et al. 1988). The resulting recombinant RNAs hybridize to their target sequences as ordinary hybridization probes do and, as in a classical hybridization assay, nonhybridized probes are then washed away. The hybridized probes are then freed from their targets and released into solution. What makes these recombinant-RNA probes particularly useful is that they can then be exponentially amplified by incubation with the RNA-directed RNA polymerase, Qxcex2 replicase (Haruna and Spiegelman, 1965). As many as 109 copies of each replicatable probe can be synthesized in a single 30-minute incubation (Lizardi, et al. 1988). Furthermore, the extreme specificity of Qxcex2 replicase for its own template RNA (Haruna and Spiegelman, 1965) assures that only the replicatable probes will be amplified. The large number of copies synthesized can easily be quantitated by incorporating radioactive nucleotides or by measuring the fluorescence of an intercalating dye such as ethidium bromide. Because as little as a single molecule of RNA can initiate exponential amplification (Levisohn and Spielelman, 1968), this approach offers the prospect of developing sensitive diagnostic assays.
The present invention provides an assay designed to detect very small amounts of HIV-1 mRNA. The assay format was designed to meet these criteria: (a) because of the desirability of developing a method that can screen a large number of samples, the selected format has to be fast and simple, thus precluding the fractionation of cells or the isolation of nucleic acids, and necessitating the use of solution hybridization; and (b) because nonhybridized probes are amplified by Qxcex2 replicase along with hybridized probes, the format must include an extremely efficient means of removing the nonhybridized probes. Hybridization is extremely efficient in solutions of the chaotropic salt, guanidine thiocyanate (Thompson and Gillespie, 1987), and concentrated solutions of guanidine thiocyanate will lyse cells, denature all proteins (including nucleases), liberate nucleic acids from cellular matrices, and unwind DNA molecules, permitting hybridization to occur without interference from cellular debris (Pelligrino, et al. 1987). The xe2x80x9creversible target capturexe2x80x9d procedure (Morrisey, et al. 1989) is an efficient means for removing nonhybridization probes. In this improved xe2x80x9csandwich hybridizationxe2x80x9d technique (Ranki, et al. 1983; Syvanen, et al. 1986), probe-target hybrids are bound to the surface of paramagnetic particles. After the particles are washed to remove nonhybridized probes, the hybrids are released from the particles, and then bound to a new set of particles for another washing. Repeating this procedure several times dramatically reduces the concentration of nonhybridized probes (Morrissey, et al. 1989).
It is imperative that sensitive tests be developed for the detection of human retroviruses. Exogenously acquired retrovirus infections have been shown to be pathogenic. Human T-lymphotropic virus type I (HTLV-I) (Poiesz et al., 1980) and type II (HTLV-II) (Kalyanaraman et al., 1982) induce the transformation and proliferation of immature T lymphocytes, causing T-cell leukemia/lymphoma; and human immunodeficiency virus type 1 (HIV-1) (Popovic et al., 1984) and type 2 (HIV-2) (Clavel et al., 1986) induce a T-cell cytopathology that depletes T4 cells, causing acquired immune deficiency syndrome (AIDS). Human retroviruses are transmitted by contaminated blood and blood products and by sexual contact (Curran et al., 1985; Fauci, 1986). In addition, children may be infected perinatally or transplacentally; and intravenous drug abusers spread infection by sharing contaminated hypodermic needles. It is thus essential that effective procedures be developed for the detection of all known pathogenic retroviruses, in order to screen donated blood and to identify asymptomatic individuals who are carriers.
This invention also concerns the development of extremely sensitive assays for the detection of blood cells infected with pathogenic retroviruses. The assays utilize novel recombinant RNAs that serve as specific hybridization probes and also serve as templates for their own exponential amplification by Qxcex2 replicase. Since more than one billion copies of each hybridized recombinant-RNA probe can be synthesized in a short incubation, extreme sensitivities can be achieved. These assays can be used to routinely screen donated blood and to identify asymptomatic individuals who are carriers, to prevent the spread of retroviral diseases.
Another method of detecting a small number of targets is the polymerase chain reaction (Saiki et al., 1985; Mullis and Faloona, 1987). In this scheme, oligonucleotide probes bind to targets and then serve as primers for DNA polymerase. Since as many as a million copies of each target region can be generated, great sensitivity can be achieved. Moreover, probes that are not bound to their targets cannot be elongated, so their presence does not generate a background signal. However, there are a number of significant disadvantages to using the polymerase chain reaction: DNA polymerase is inhibited by many components of the sample. For example, hemoglobin interferes with amplification; consequently, peripheral blood mononuclear cells must be separated from other cells prior to amplification. Alternatively, cellular DNA is isolated prior to analysis. Although greater sensitivity can be achieved by detecting retroviral messenger RNA, DNA polymerase cannot copy RNA, necessitating an additional reverse transcription step. Furthermore, each cycle of amplification involves incubation at two different temperatures, necessitating the use of a relatively expensive xe2x80x9ctemperature cyclerxe2x80x9d; and the 20 or more cycles needed for each assay consume time. The amount of DNA that can be synthesized with a given pair of primers is apparently limited by unidentified factors in the reaction to approximately one million copies of each target. Furthermore, when several primer pairs are used in a single assay (to detect different targets simultaneously), the amplification of each target is suppressed, resulting in markedly lower yields. Finally, the amplified DNA products frequently include a variety of unrelated background DNAs, necessitating an electrophoretic analysis or an additional hybridization to identify the desired DNA (Abbott et al., 1988). Although a variety of schemes exist to surmount these drawbacks, practical assays are likely to be rather cumbersome, time-consuming, and expensive.
In Kramer, et al., U.S. Pat. No. 4,786,600, issued Nov. 22, 1988, there are disclosed replicatable recombinant single-stranded RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis by the polymerase and a heterologous sequence of interest derived from a different RNA molecule inserted at a specific site in the internal region of the recombinant molecule. Kramer, et al. does not teach or suggest that if the inserted sequence is a hybridization probe sequence, that the resulting molecules can be replicated after hybridization to produce multiple copies for detection.
In Chu, et al., U.S. application Ser. No. 852,692, filed Apr. 16, 1986, methods are disclosed for determining the presence of targets, i.e., analytes, by linking a replicatable RNA, which serves as a reporter group, to a probe, e.g., an oligonucleotide, an antibody or lectin. Chu, et al. also disclose that an RNA-directed RNA polymerase can then be used after hybridization has occurred to produce multiple copies of the replicatable RNA for detection. However, Chu, et al. do not describe a method in which different recombinant-RNA xe2x80x9cprobexe2x80x9d sequences can be used simultaneously in the same assay.
Diagnostic assays that use Qxcex2 replicase to exponentially amplify a replicatable RNA reporter have many advantages: Qxcex2 replicase is highly specific for its own template RNA (Haruna and Spiegelman, 1965c), and will not copy any other RNA in the sample. Amplification can be initiated with as little as one molecule of RNA (Levisohn and Spiegelman, 1968). Incubations are carried out at 37xc2x0 C. and take less than 30 minutes; and the large amount of RNA that is synthesized (typically, 200 nanograms in a 50 microliter reaction) enables its detection by simple calorimetric methods. Quantitation of the number of targets originally present in a sample can occur over a range of target concentrations that exceeds 1,000,000-fold (Lizardi et al., 1988). The materials required for these assays are inexpensive, and the simplicity of the procedure lends itself to automation. Protocols that permit the simultaneous detection of cells infected with different pathogenic retroviruses in the same sample are effective with the recombinant RNA reporters of the subject invention.
The present invention concerns a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising (a) a recognition sequence for the binding of an RNA-directed RNA polymerase; (b) a sequence required for the initiation of product strand synthesis by the polymerase; and (c) a heterologous RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo- or polynucleotide of interest.
The invention also provides a method for determining the presence or concentration of an oligo- or polynucleotide of interest in a sample, comprising the steps of: (a) forming a specific complex between the recombinant-RNA probe molecule described above and the oligo- or polynucleotide of interest, by incubating the sample with the recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the recombinant-RNA probe molecules that are hybridized to the oligo- or polynucleotide of interest; and (d) detecting the recombinant-RNA probe molecules synthesized in step (c), thereby determining the presence or concentration of the oligo- or polynucleotide of interest.
The invention herein further provides a method for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample, comprising the steps of: (a) forming specific complexes between a mixture of different types of recombinant-RNA probe molecules described above, each type having a different inserted sequence, and the oligo- or polynucleotides of interest, by incubating the sample with the mixture of recombinant-RNA probe molecules under suitable conditions and for a sufficient period of time to permit complementary nucleotide sequences to hybridize; (b) removing unhybridized recombinant-RNA probe molecules from the reaction mixture; (c) incubating the reaction mixture with an RNA-directed RNA polymerase capable of synthesizing additional copies of the recombinant-RNA probe molecules which are hybridized to the oligo- or polynucleotides of interest; (d) separating the mixture of synthesized recombinant-RNAs by hybridizing them to an ordered array of polynucleotides bound to a solid support where each of the polynucleotides is complementary to one type of synthesized recombinant-RNA; and (e) detecting the recombinant-RNA probe molecules produced in step (d), thereby determining the presence or concentration of each oligo- or polynucleotide of interest.